抗松针褐斑病湿地松胚性愈伤组织的超低温保存

Cryopreservation of Embryogenic Callus of Pinus elliottii with Resistance to Brown Spot Needle Blight

本实验以抗松针褐斑病湿地松胚性愈伤组织27-1为材料,运用正交实验方法探究其超低温冷冻保存方法。结果表明:预培养时间、二甲基亚砜(DMSO)浓度、蔗糖浓度、聚乙二醇(PEG) 8000浓度和解冻温度对解冻后愈伤组织的活性均有显著影响。结合解冻后愈伤组织活力及其恢复生长情况,得出效果较好的超低温保存方法为:继代培养10~12 d生长状态良好的愈伤组织,添加0.5 mol/L蔗糖的高渗培养基预培养36 h;5%DMSO,15%PEG 4000,0.5 mol/L蔗糖3种冷冻保护剂组合使用,程序降温至-80℃停留30 min后投入液氮罐中保存;32℃水浴解冻,蔗糖溶液清洗后转接至LP培养基上恢复生长。在此程序处理下,冷冻90 d的愈伤组织1个月后可恢复再生,再生率最高达100%,再生愈伤组织生长状态良好并依然保持分化成熟体胚的能力。

This study was carried out with embryogenic callus called 27-1 of disease-resistant Pinus elliottii,and cryopreservation program was explored by using the orthogonal experiment method.The results showed that preculture time,concentration of dimethyl sulfoxide(DMSO),concentration of sucrose,concentration of polyethylene glycol(PEG) 8000 and thawing temperature dramatically affected the vitality of callus after thawing.Combined with activity and recovery of callus after thawing,the method of cryopreservation with better effect was obtained:the callus in a good growth state which cultured for 10~12 days after subculture,was pre-cultured in the hypertonic medium with 0.5 mol/L sucrose for 36 h;and then pretreated in buffer of 5% DMSO,15% PEG 4000 and 0.5 mol/L sucrose,cooled to-80℃ and preserved in liquid nitrogen after maintained at-80℃ for 30 min;thawed at 32℃,sucrose solution was cleaned and transferred to LP medium to resume growth.Under this procedure,callus froze for 90 days can be recovered and regenerated after 1 month with a regeneration rate up to 100%.The regenerated callus grows well and still maintains the ability to differentiate into mature somatic embryos.